聚合酶链反应检测疱疹Ⅱ型病毒先天性宫内感染的研究

以人工合成的单纯疱症Ⅱ型病毒DNA多聚基因片段为引物，采用聚合酶链反应技术(PCR)检查早孕绒毛标本中疤疹I型病毒感染，以80例自发和人工流产的早孕绒毛组织标本进行基因扩增，31例绒毛标本中检出HSV—Ⅱ，并与同一绒毛标本的免疫组织化学结果进行比较，PCR技术免除了昂贵繁杂的分子杂交过程，具有极高的敏感和特异性，为早期、快速产前诊断HSV先天性宫内感染的最佳技术。     

单纯疱疹病毒Ⅰ型糖蛋白B基因疫苗的构建及细胞免疫学研究

目的：构建、制备单纯疱疹病毒Ⅰ型(HSV-Ⅰ)糖蛋白BDNA疫苗，检测其诱导机体产生的细胞免疫应答。方法：PCR扩增编码HSV-Ⅰ gB去除N端部分信号肽序列(39bp)的基因片段(2673bp)，定向插入pcDNA3载体中，构建pcDNA3-gB基因疫苗并对其进行PCR、酶切及测序鉴定。于BALB／c小鼠注射免疫3次，观察小鼠CD4^ 、CD8^ T细胞亚群的变化，CFSE／PI双标记的流式细胞计数法检测CTL活性。结果：双酶切分析结果为目的基因(2．7kb)和线性质粒pcDNA3(5．4kb)；PCR扩增结果为特异产物(2．7kb)；测序结果与GenBank gB基因序列同源性达99．5％；CTL，活性增强；BALB／c小鼠脾CD4^ T细胞增加。结论：经鉴定证实了HSV-Ⅰ gB基因疫苗的构建；HSV-Ⅰ gB基因疫苗可以诱导较强的细胞免疫应答，应用于预防HSV-Ⅰ的感染具有良好的前景。     

单核巨噬细胞对HSV－1的抵抗及LPS、BCG对其的影响

以ＨＳＶ－１接种人单核细胞系Ｕ９３７和小鼠腹腔巨噬细胞，通过病毒感染滴度测定，间接荧光抗体试验检测病毒抗原及多聚酶链反应检测病毒基因，在体外实验中研究了单核巨噬细胞对ＨＳＶ－１的抗性及其影响因素。结果证实，Ｕ９３７和小鼠腹腔巨噬细胞均能逐步清除ＨＳＶ－１，从感染中恢复；１０～２０μｇ／ｍｌＬＰＳ处理细胞能削弱细胞的抗性、促进病毒基因表达，而５μｇ／ｍｌＢＣＧ能增强细胞的抗性、加速病毒的灭活和清除。     

Local immune responses and systemic cytokine responses in zoster: relationship to the development of postherpetic neuralgia

Varicella zoster virus (VZV) causes varicella (chickenpox) as the primary infection and zoster (shingles) on reactivation from latency, often many years later. One of the most common and most severe sequela of zoster is postherpetic neuralgia (PHN). Apart from age, factors which predispose towards PHN are unknown. In the present study, the concentration of a variety of Th1 and Th2 cytokines in the serum of 30 zoster patients at the time of the acute disease were correlated with the subsequent development of PHN in nine of these patients, but no association was found. In addition, although some cytokines such as IFN-gamma, IL-6 and IL-8 were slightly raised in the zoster group compared with a group of normal healthy subjects of a similar age distribution, these differences only verged on significance. Antibody titres to VZV were raised in the zoster group compared with the controls but these did not differ between the patients who developed PHN and those who did not. Biopsies of zoster lesions were collected from nine patients. There were significantly fewer infiltrating lymphocytes in the lesions of the three patients who subsequently developed PHN compared with the six who did not, although the expression of the neuropeptide, substance P, did not differ between the two groups. It is possible that the poor inflammatory response at the time of the acute zoster may result in less effective containment of the VZV and more damage in the dermatome, thus contributing to the persistence of the neuralgia.     

Detection of herpesvirus cervicovaginitis by a sequential Papanicolaou--immunoperoxidase technique

Herpes simplex virus (HSV) infection constitutes an immediate threat to the neonate of pregnant women who deliver vaginally, and thus requires a rapid, specific means of diagnosis that is easily applicable to cervicovaginal smears. We applied the peroxidase-antiperoxidase technique to variously fixed, previously stained Papanicolaou smears from 60 women with HSV and 18 negative controls using an HSV-2 antibody and either diaminobenzidine (DAB) or aminoethylcarbazol (AEC) as the chromogen and Mayer's, Gill's, or Lillie-Mayer's hematoxylin as the counterstain. With Papanicolaou stain alone, there was adequate cytologic demonstration of either single cells in aggregates (11%), syncytial giant cells (40%), or both (49%) that displayed a ground-glass appearance (68%), discrete nuclear inclusions (5%), or both (28%). With the peroxidase-antiperoxidase technique, 57 of the 60 HSV specimens (95%) stained moderately or strongly positive for HSV-2, as did sections of herpetic encephalitis and esophagitis. There was no false-positive staining in any of the 18 control smears revealing koilocytosis, Chlamydia, or nonspecific vaginitis. Positivity of the immunostain was more vivid and evenly dispersed through-out the cytoplasm with AEC than with DAB, but tended to wash away with alcohol-based counterstaining. In contrast, DAB was more stable, but was positive predominantly at the cell periphery and cytoplasmic processes. Lillie-Mayer's stain provided the best counterstaining for the cytologic visualization of virocytes and accompanying inflammatory and epithelial cells, which revealed a minimal degree of atypia. The fixative used had no influence on the frequency or degree of immunopositivity of virocytes, but wet fixation with 95% alcohol or Carbowax led to less background staining than Spray-Cyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

鼻咽癌患者树突状细胞对EBV特异性细胞毒T细胞的免疫反应

目的：用负载EB病毒抗原LMP-2表位肽段的鼻咽癌患者树突状细胞（DC）诱导自身CD8^＋T细胞，观察其细胞毒性T细胞的免疫应答能力。方法：在体外分离并诱导成熟HLA—A2基因型鼻咽癌患者自身的DC，分别负载EB病毒抗原LMP-2的HIA—A2限制型两个表位肽段CLGGLLTMV（CLG）和LTAGFIFL（LTA）后，诱导自身CD8^＋T细胞，培养2周后，运用酶联免疫斑点法（Elispot）和HLA-肽四聚体法（Tetramer）检测诱导后T细胞的免疫应答情况。结果：经DC抗原呈递后分泌IFN-γ的CLG和LTA特异性CD8^＋T细胞数，分别为（42．67±33．79）个／孔和（25．67±18．25）个／孔，而呈递前分别为（2．67±1．97）个／孔和（5．33±1．86）个／孔。两者IFN-γ^＋CD8^＋T细胞数均有明显增加（P〈0．05）。CLG和LTA肽段特异性细胞毒性T细胞（CTL）的中位数在呈递前分别为0．135％和1．14％；呈递后则为1．045％和1．945％。两者较呈递前均明显增加（P〈0．05）。结论：负载EB病毒抗原LMP-2表位肽段的DC诱导鼻咽癌患者自身CD8^＋T细胞，可产生特异性CTL，对鼻咽癌患者的免疫治疗具有潜在的应用价值。     

HSV-TK/GCV自杀基因系统治疗宫颈癌的研究

目的探讨HSV-TK/GCV自杀基因系统对人宫颈癌细胞系Hela体外及体内杀伤作用及其产生的旁观者效应.方法采用脂质体转染法将GINaTK载体转入包装细胞PA317.取病毒上清液感染人宫颈癌细胞Hela,得到带有HSV-TK基因的Hela/TK细胞,并将其分别用于体外和体内实验.结果载体HSV-TK导入了PA317细胞.体外实验结果显示,当Hela/TK细胞数占混合细胞10%时,低浓度(10μg/ml)的GCV就可将50%左右的肿瘤细胞杀死.体内实验结果显示GCV可明显抑制Hela/TK细胞在BALB/C小鼠体内的肿瘤形成.经GCV治疗后,肿瘤体积分别较对照组肿瘤体积缩小约11.1%、30.6%和47.2%(均P＜0.001);RT-PCR检测HSV-TK基因在肿瘤组织中有表达;实验组肿瘤组织与对照组相比存在明显的病理学改变.结论逆转录病毒可介导HSV-TK基因转入人宫颈癌细胞Hela并获稳定表达,HSV-TK/GCV自杀基因系统在体内外对宫颈癌细胞均有杀伤作用,且存在明显的旁观者效应.     

Kaposi’s sarcoma following malignant mesothelioma

We report the unusual occurrence of Kaposi’s sarcoma following asbestos-related malignant mesothelioma, in a human deficiency virus (HIV)-negative Italian man. Seropositivity to human herpes virus 8 (HHV8) was documented at the time of mesothelioma diagnosis and preceded the onset of Kaposi’ sarcoma with a time lapse of 13 months. HHV8 DNA was detected by polymerase chain reaction in lesional Kaposi’s sarcoma but not within mesothelioma. By immunostaining, mesothelioma cells expressed interleukin-6 and platelet-derived growth factor, which are important for survival of Kaposi’s sarcoma cells. Besides the possibility of a casual association, we hypothesize that mesothelioma-linked factors may have contributed to the development of Kaposi’sarcoma in the presence of HHV8 infection. Received: 12 April 1999 / Accepted: 24 June 1999     

表达单纯疱疹病毒Ⅱ型糖蛋白D的重组痘苗病毒活疫苗株的建立

我们以前曾报道，表达单纯疱疹病毒Ⅱ型糖蛋白Ｄ（ＨＳＶ－２ｇＤ）的重组痘苗病毒（实验疫苗株）能保护被免疫小鼠抵抗致死量ＨＳＶ－２病毒的攻击。在此工作基础上，严格按人用疫苗研究要求的实验条件，成功地建立了表达ＨＳＶ－２ｇＤ的重组痘苗病毒活疫苗株。首先将经聚合酶链反应（ＰＣＲ）修饰的ＨＳＶ－２ｇＤ基因插入痘苗表达质粒ｐＪＳＢ１１７５，置于痘苗病毒Ｐ７５Ｋ早／晚期启动子控制下。将此重组质粒用Ｌｉｐｏｆｅｃｔｉｎ方法转染已受野型ＴＫ＋痘苗病毒天坛７６１株感染的人胚肺二倍体细胞。经同位素探针（３２Ｐ－ＨＳＶ－２ｇＤ）原位杂交法和３轮蚀斑纯化，筛选出基因组内整合有ＨＳＶ－２ｇＤ基因的重组痘苗病毒。斑点和Ｓｏｕｔｈｅｒｎ杂交证实，ＨＳＶ－２ｇＤ基因已插入痘苗病毒基因组内预期的ＴＫ区段，间接免疫荧光检测显示，重组病毒感染细胞后能有效地表达ＨＳＶ－２ｇＤ蛋白。     

Immunohistochemical study of skin lesions in herpes zoster

Summary Thirty-seven biopsy skin tissues of herpes zoster taken from 27 patients were analysed immunohistochemically using two monoclonal antibodies detecting either nucleocapsid or glycoproteins of varicella-zoster virus (VZV) on paraffin sections of formalin fixed tissues. Skin lesions of herpes zoster were divided clinically into four stages: erythematous, vesicular, pustular and ulcerative. In the erythematous stage, VZV antigens, if detected, were found only within ballooning cells in the lower epidermis or follicular epithelium. In the vesicular stage, antigens were detected in the cells around and within the intraepidermal vesicles and in histiocytes or fibrocytes of the dermis in all cases and in the endothelial or perineural cells in 10 of 14 cases. In the pustular stage, the antigens were observed in degenerated or necrotic keratinocytes and multinucleated giant cells within pustules and some necrotic cells in the dermis. In the ulcerative stage, the viral antigens were detected only at the ulcer margin and around the hair shaft in 2 of 7 cases. These results suggest that VZV initially involves the epidermis in the erythematous stage, subsequently invades the dermis in the vesicular stage, and disappears in the early ulcerative stage.